Electrophysiologic Effects of Beta-blocking Agent, Tilisolol, on Isolated Guinea Pig Ventricular Myocytes

BACKGROUND: Electrophysiologic effects of a beta-blocking agent, tilisolol, were studied with isolated guinea pig ventricular myocytes using the whole cell patch clamp technique. METHODS AND RESULTS: Tilisolol at 10 μM or higher concentrations prolonged action potential duration (APD) at 90% repolarization (APD(90)) and at 100 μM or higher concentrations shortened APD at 20% repolarization (APD(20)) without changes in resting membrane potential. At 10 μM concentration tilisolol prolonged APD(90) from 236.6 +/- 55.3 ms in the control to 253.4 +/- 52.4 ms (n = 16; P <.01), while APD(20) was unaffected. At 100 μM tilisolol, APD(20) was shortened from 143.6 +/- 15.7 ms in the control to 133.7 +/- 22.6 ms (n = 8; P <.05). Under voltage clamp, tilisolol decreased the delayed rectifier K(+) current (I(K1)). Applications of 10 μM and 100 μM tilisolol reduced the maximal conductance of I(K) by 35.7 +/- 3.5% and 47.4 +/- 3.5% of the control, respectively, without changes in voltage dependence (n = 10). Tilisolol at 100 μM decreased the L-type Ca(2+) current (I(Ca.L)) by 22.0 +/- 9.8% (n = 6) of the control, and the inactivation curve was shifted to a hyperpolarizing direction. CONCLUSIONS: Tilisolol has a direct membrane action to depress I(K) and I(Ca.L), in addition to its beta-receptor blocking action.     

血管紧张素Ⅱ诱导体外培养新生大鼠心肌细胞凋亡的研究

本实验旨在研究AngⅡ作用下 ,心肌细胞凋亡的情况。取新生SD大鼠的左心室组织 ,酶解分散成单细胞 ,2 0 %小牛血清培养基中培养 8天。更换培养基 ,加入AngⅡ。细胞在 10 -9M的AngⅡ下作用 2 4小时。以低张荧光PI染液染色 ,用流式细胞术分析G1期前低二倍体的凋亡细胞。结果显示AngⅡ组凋亡率为 16 2 9%±4 3 1% (n =8) ,对照组凋亡率为 5 0 4%± 2 72 % (n =8) ,P <0 0 0 1,有显著性差异。提示AngⅡ作用 2 4小时后引起体外培养的心肌细胞发生凋亡。     

Angiotensin Converting Enzyme and Angiotensin II Type 1 Receptor Polymorphisms in Patients with Coronary Aneurysms

BACKGROUND: Conflicting results have been reported regarding the association of gene polymorphisms in the renin-angiotensin system (RAS) with different aspects of coronary artery disease (CAD), such as myocardial infarction, neointimal hyperplasia or coronary artery vasomotion. Since previous studies have linked angiotensin II to aneurysmal disease, our study hypothesis was that RAS gene polymorphisms may be associated with aneurysm remodeling in response to CAD. METHODS: The study population was selected from a series of 3862 consecutive patients who underwent coronary angiography in our institution. One hundred and thirteen consecutive patients with at least one coronary aneurysm (CA) were compared to 226 randomized control patients without CA. DNA was extracted from white blood cells. The angiotensin-converting enzyme (ACE) I/D and angiotensin type 1 receptor (AT1-R) A/C polymorphisms were detected using previously published techniques. RESULTS: The distributions of the three ACE genotypes were similar in both groups: CA: 13%, 46%, and 41% for II, ID, and DD respectively; controls: 18%, 41%, and 41% for II, ID, and DD respectively, p = 0.45. The distributions of the three AT1-R genotypes were also similar in both groups: CA: 54%, 41%, and 5% for AA, AC, and CC respectively; controls: 55%, 33%, and 12%, for AA, AC, and CC respectively, p = 0.08. CONCLUSION: Our results provide further information on the role of RAS polymorphisms on specific mechanisms implicated in CAD. Although an activated RAS may theoretically promote aneurysm formation, the 2 RAS polymorphisms analyzed in this study are not associated with this process in coronary arteries.     

Solubilized Receptor for Intrinsic Factor-Vitamin B12 Complex from Guinea Pig Intestinal Mucosa

the absorption of vitamin B(12) in many animals requires its prior association with intrinsic factor (IF) and attachment to a specific receptor in the intestine. Employing Triton X-100, we have solubilized from guinea pig ileum a factor that binds intrinsic factor-vitamin B(12) complex (IF-B(12)). This binding factor was soluble to the extent that it was not sedimented by centrifugation at 100,000 g for 1 h and was small enough to enter the included volume of a Sepharose 4-B column. Furthermore, the ileal extract contained no microfine particles of membrane upon electron microscopic search. When a portion of the extract was incubated with a mixture of gastric juice and (57)Co-labeled vitamin B(12), a portion of the radioactivity was excluded from a Sephadex G-200 column. When gastric juice from a patient with a congenital abnormality of IF that prevented its binding to intestine was substituted for normal human gastric juice, radioactivity was not excluded from the gel, indicating failure of this abnormal IF-B(12) to bind to the intestinal extract. These data suggested the presence of a specific binder of IF-B(12) in the ileal mucosal extract. The reactions of normal IF-B(12) with the solubilized binding factor and with the membrane-bound "receptor" had several characteristics in common, including calcium dependence, temperature independence, and pH optimum near neutral. Extracts from the distal intestine showed more activity than did those from the proximal. The solubilized binding facter seemed specific for IF-B(12) in that it was not blocked by prior incubation with excesses of either free vitamin B(12) or IF. Binding activity of the extract was decreased by incubation at pH 2.0, by heating to 56 degrees C, and by incubation with chymotrypsin and dithiothretiol. Incubation with trypsin, neuraminidase, and sulphydryl blockers did not affect it.The Triton X-100 extract of guinea pig ileal mucosa contains a specific binding factor that probably is the receptor for IF-B(12). This appears to be a protein with function dependent on peptide and disulphide linkages.     

The Adrenal Receptor for Angiotensin II is Altered in Essential Hypertension

To determine the mechanism underlying altered adrenal responsiveness in patients with essential hypertension, the renin-angiotensin-aldosterone axis was assessed in normotensive and hypertensive subjects using three pharmacological probes: SQ 20881, a converting enzyme inhibitor; saralasin, a competitive angiotensin antagonist with prominent agonist properties; and angiotensin itself. All subjects were studied while supine and in balance on a 10 meq Na/100 meq K intake. The decrement in plasma aldosterone with SQ 20881 in 26 hypertensive subjects (15+/-3 ng/dl) was normal (13+/-4 ng/dl), suggesting that the altered adrenal responsiveness in hypertensives is not because of a change in a postreceptor event or in the relative contribution of angiotensin to the control of aldosterone secretion.Saralasin at a dose (0.1 mug/kg per min) that reduced aldosterone levels in all normals produced a normal aldosterone decrement (14+/-3 ng/dl) in 19 patients with renovascular hypertension (12+/-4 ng/dl). The same dose, however, had no net effect on plasma aldosterone levels in 70 patients with normal or high renin essential hypertension (-1+/-1 ng/dl) despite identical metabolic balance and control renin and angiotensin levels. The altered response could be explained by an agonist effect, aldosterone rising in 45 of the essential hypertensives. There were no significant differences between normal and abnormal responders in pre- and postcortisol, -potassium, -renin and -angiotensin concentrations.Angiotensin was infused (0.1-3 ng/kg per min) in 15 patients with normal renin essential hypertension, previously studied with saralasin. A probit transformation defined the dose required to induce a 50% increase in aldosterone (ED50). In the patients in whom aldosterone rose with saralasin, the dose required to induce a 50% increase was significantly greater (P < 0.001) than in those in whom aldosterone fell normally (1.02+/-0.06 [SD] vs. 0.38+/-0.07 ng/kg per min). Vascular responses were similar in the various groups. We conclude that altered adrenal responsiveness to angiotensin in some essential hypertensive patients is secondary to a change in the interaction of angiotensin with its adrenal receptor.     

Left Ventricular Ejection Fraction and Absence of ACE Inhibitor/Angiotensin II Receptor Blocker Predicts Appropriate Defibrillator Therapy in the Primary Prevention Population

Introduction: Implantable cardioverter defibrillators (ICD) significantly reduce mortality in patients with left ventricular (LV) dysfunction. However, little is known of the predictors of appropriate device activation in the primary prevention population. The aim of the present study was to determine predictors of appropriate device therapy in patients receiving ICDs for primary prevention. Methods & Results: One hundred twenty‐six patients with a left ventricular ejection fraction (LVEF) of < 35% and no prior documented ventricular arrhythmias underwent ICD implantation. The ICD implanted was single chamber in 60 (48%), dual chamber in 10 (8%), and biventricular in 56 (44%) patients and programmed with a single ventricular fibrillation (VF) zone at >180 beats per minute. Mean age was 58 ± 13 years and mean LVEF was 23 ± 7%. Fifty‐two percent had ischemic cardiomyopathy and 66% were New York Heart Association heart failure class II/III. During a mean follow‐up period of 589 ± 353 days, 17 (13%) patients received appropriate device therapy and three (4%) received inappropriate shocks. Appropriate ICD therapy was associated with reduced LVEF (mean 19.9% vs 23.7%, P = 0.02) and the patients were less likely to have received angiotensin‐converting enzyme inhibitor (ACEI) or angiotensin II receptor blockers (AIIRB) (65% vs 90%, P = 0.04). Multivariate analysis revealed lack of ACEI/AIIRB (odds ratio [OR]= 0.06, 95% confidence interval [CI]= 0.01–0.37, P = <0.01) and lower LVEF (OR = 0.88, 95% CI 0.79–0.98, P = 0.02) predicted appropriate device activation. There was no difference in transplant‐free survival between the appropriate therapy and no/inappropriate therapy groups, LVEF <20% and LVEF >20% group, and lack of ACEI/AIIRB and ACEI/AIIRB group. Conclusion: Appropriate device activation occurred in 13% of patients in a primary prevention population. LVEF and absence of ACEI/AIIRB predicted appropriate ICD therapy. (PACE 2010; 33:696–704)     

Angiotensin II in septic shock

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2015 and co-published as a series in Critical Care. Other articles in the series can be found online at http://ccforum.com/series/annualupdate2015. Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901.     

Angiotensin II in septic shock

Septic shock is a life threatening condition and a medical emergency. It is associated with organ dysfunction and hypotension despite optimal volume resuscitation. Refractory septic shock carries a very high rate of mortality and is associated with ischemic and arrhythmogenic complications from high dose vasopressors. Angiotensin II (AT-II) is a product of the renin-angiotensin-aldosterone system. It is a vasopressor agent that has been recently approved by FDA to be used in conjunction with other vasopressors (catecholamines) in refractory shock and to reduce catecholamine requirements. We have reviewed the physiology and current literature on AT-II in refractory septic/vasodilatory shock. Larger trials with longer duration of follow-up are warranted to address the questions which are unanswered by the ATHOS-3 trial, especially pertaining to its effects on lungs, brain, microcirculation, inflammation, and venous thromboembolism risk.     

Angiotensin II in organ organopathy

Angiotensin II induces the organ derangements is not generated by the so-called classic rennin-angiotensin system but by the tissue angiotensin II generating system. We have confirmed this evidences by using different types of animal models such as hypertension, arteriosclerosis, vascular narrowing by balloon injury and in vein graft disease. In addition, we found that the ACE and chymase activities are increased in human aneurismal aorta. ACE inhibitor is effective to protect the development of hypertension and arteriosclerosis, but not to other models because the angiotensin II produced by chymase is involved in such diseases. Angiotensin II produced separately by ACE and chymase, participates independently in the development of vascular derangements.     

Discontinuation of Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blockers in Chronic Kidney Disease

ObjectiveTo assess the patterns of angiotensin converting enzyme inhibitors and angiotensin receptor blockers (ACE-I/ARB) discontinuation in the setting of chronic kidney disease (CKD) progression in real-world clinical practice.Patients and MethodsWe identified incident ACE-I/ARB users with a baseline estimated glomerular filtration rate (eGFR) ≥15 mL/min/1.73 m² and without end-stage renal disease in the Geisinger Health System between January 1, 2004, and December 31, 2015. We investigated the associations of CKD stage, hospitalizations with and without acute kidney injury (AKI), serum potassium, bicarbonate level, thiazide, and loop diuretic use with ACE-I/ARB discontinuation.ResultsAmong the 53,912 ACE-I/ARB users, the mean age was 59.9 years, and 50.6% were female. More than half of users discontinued ACE-I/ARB within 5 years of therapy initiation. The risk of ACE-I/ARB discontinuation increased with more advanced CKD stage. For example, patients who initiated ACE-I/ARB with CKD stage G4 (eGFR: 15-29 mL/min/1.73 m²) were 2.09-fold (95% CI, 1.87-2.34) more likely to discontinue therapy than those with eGFR ≥ 90 mL/min/1.73 m². Potassium level greater than 5.3 mEq/L, systolic blood pressure ≤ 90 mm Hg, bicarbonate level < 22 mmol/L, and intervening hospitalization—particularly AKI-related–were also strong risk factors for ACE-I/ARB discontinuation. Thiazide diuretic use was associated with lower risk, whereas loop diuretic use was associated with higher risk of discontinuation.ConclusionIn a real-world cohort, discontinuation of ACE-I/ARB was common, particularly in patients with lower eGFR. Hyperkalemia, hypotension, low bicarbonate level, and hospitalization (AKI-related, in particular) were associated with a higher risk of ACE-I/ARB discontinuation. Additional studies are needed to evaluate the risk–benefit balance of discontinuing ACE-I/ARB in the setting of CKD progression.     

Angiotensin II in experimental hyperdynamic sepsis

Introduction  

Angiotensin II (Ang II) is a potential vasopressor treatment for hypotensive hyperdynamic sepsis. However, unlike other vasopressors, its systemic, regional blood flow and renal functional effects in hypotensive hyperdynamic sepsis have not been investigated.     

Comparative Efficacy of Angiotensin II Type 1 Receptor Blockers Against Ventilator‐Induced Diaphragm Dysfunction in Rats

Mechanical ventilation (MV) is a life‐saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (termed ventilator‐induced diaphragm dysfunction (VIDD)). Although VIDD is a major risk factor for problems in weaning patients from MV, a standard therapy to prevent VIDD does not exist. However, emerging evidence suggests that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the essential characteristics of AT1R blockers (ARBs) required to protect against VIDD remain unclear. To determine the traits of ARBs that are vital for protection against VIDD, we compared the efficacy of two clinically relevant ARBs, irbesartan and olmesartan; these ARBs differ in molecular structure and effects on AT1Rs. Specifically, olmesartan blocks both angiotensin II (AngII) binding and mechanical activation of AT1Rs, whereas irbesartan prevents only AngII binding to AT1Rs. Using a well‐established preclinical model of prolonged MV, we tested the hypothesis that compared with irbesartan, olmesartan provides greater protection against VIDD. Our results reveal that irbesartan does not protect against VIDD whereas olmesartan defends against both MV‐induced diaphragmatic atrophy and contractile dysfunction. These findings support the hypothesis that olmesartan is superior to irbesartan in protecting against VIDD and are consistent with the concept that blockade of mechanical activation of AT1Rs is a required property of ARBs to shield against VIDD. These important findings provide a foundation for future clinical trials to evaluate ARBs as a therapy to protect against VIDD.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Prolonged mechanical ventilation results in ventilator‐induced diaphragm dysfunction (VIDD). This is significant because VIDD is a major risk factor for problems in weaning patients from the ventilator. Currently, no standard treatment exists to prevent VIDD. However, emerging evidence reveals that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the specific properties of angiotensin receptor blockers (ARBs) required to protect against VIDD remain unknown.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ What characteristics of ARBs are vital for protection against VIDD?

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ ARBs that prevent angiotensin II binding to AT1Rs alone do not protect against VIDD. In contrast, ARBs that block mechanical activation of AT1Rs are protective against VIDD.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ These new findings provide a foundation for future testing of ARBs in clinical trials.

Mechanical ventilation (MV) is used annually in > 13 million patients in the intensive care unit. ¹ Although MV is a life‐saving intervention for many critically ill patients, an unintended consequence of prolonged MV is the rapid development of inspiratory muscle (i.e., diaphragm) weakness. This MV‐induced diaphragmatic weakness is due to both atrophy and contractile dysfunction of the diaphragm, collectively known as ventilator‐induced diaphragm dysfunction (VIDD). ² VIDD is clinically important because diaphragmatic weakness is a major risk factor for problems in “weaning” patients from the ventilator. ³ This failure to wean results in extended time on the ventilator along with increased morbidity and mortality. ⁴ Currently, no standard therapy exists to protect against VIDD.Recent studies into the pathogenesis of VIDD reveal that the renin angiotensin system is a potential therapeutic target to prevent VIDD. Specifically, treatment with the angiotensin II type 1 receptor (AT1R) blocker (ARB) losartan protects against VIDD. ⁵ Although this study reveals that ARBs are protective against VIDD, several classes of ARBs exist and the characteristics of ARBs that provide optimal protection against VIDD remain unknown. In principle, pharmacological blockade of AT1Rs via losartan can avert VIDD by one of three mechanisms. ⁶ , ⁷ , ⁸ First, losartan prevents activation of the classical RAS pathway by blocking angiotensin (AngII) binding to AT1Rs. Second, mechanical stress can activate AT1Rs and losartan blocks mechanical activation of AT1Rs. Indeed, it is feasible that mechanical activation of AT1Rs occurs during MV due to the mechanical stress placed on the receptor due to the passive, repetitive shortening/lengthening cycles of diaphragm fibers during ventilator support. Last, it is possible that losartan protects against VIDD by a combination of blocking both AngII binding and mechanical activation of the AT1R.Prior to clinical trials, additional preclinical studies are required to determine the characteristics of ARBs that are required to protect against VIDD; this forms the rationale for the current study. To delineate the properties of ARBs needed for protection against VIDD, we tested the efficacy of two US Food and Drug Administration (FDA) approved ARBs (i.e., olmesartan and irbesartan) that differ in both molecular structure and effects on the AT1R. Olmesartan blocks both AngII binding and mechanical activation of AT1Rs whereas irbesartan blocks only ligand binding to the receptor. We hypothesized that compared with irbesartan, olmesartan will provide superior protection against VIDD.     

Intracellular Potassium Activity in Guinea Pig Papillary Muscle during Prolonged Hypoxia

During prolonged hypoxia, intracellular potassium concentration, [K](i) has been reported to fall by 70% with a concomitant decrease in the calculated potassium equilibrium potential, E(K). Nevertheless, resting membrane potential, V(m), declined only slightly. Because V(m) depolarized very little in relation to the calculated E(K), it was hypothesized that electrogenic Na-K pumping contributed up to 40 mV to V(m) during prolonged hypoxia. To further test this hypothesis we studied what changes prolonged hypoxia makes in the thermodynamically active fraction of cellular potassium, intracellular potassium activity, alpha(K) (i), and how change in alpha(K) (i) affects the relationship between V(m), E(K) and, by inference, the Na-K pump. Using double-barrel K-selective electrodes, V(m) and alpha(K) (i) were measured in quiescent guinea pig right ventricular papillary muscles superfused for 8 h with hypoxic Tyrode's solution. Over the 8-h period both V(m) and alpha(K) (i) decreased. However, the decline in V(m) was paralleled by a decrease in the E(K) calculated from alpha(K) (i). At no time was there hyperpolarization of V(m) beyond E(K).After 8 h the Na-K pump was inhibited by exposing the muscles to 0.1 mM ouabain. The onset of an increase in extracellular potassium activity, measured with a double-barrel electrode, was used to mark the amount of depolarization of V(m) due solely to pump inhibition. After hypoxia, V(m) depolarized 8.4+/-4.4 mV before extracellular potassium activity (alpha(K) (e)) increased. Thus, the decrease in alpha(K) (i) during hypoxia is much less than that reported for [K](i). The parallel decline in V(m) and E(K) and the small depolarization of V(m) with ouabain suggest that after prolonged hypoxia the Na-K pump continues to contribute to V(m), but the amount of this contribution is substantially less than previously reported.     

山莨菪碱对兔心室肌细胞离子通道电流的影响

目的研究山莨菪碱对兔正常离体心室彤睫田胞离子通道电流的影响,探讨其抗心律失常的细胞学离子机制。方法以酶解的方法分离兔心室肌外膜单个心室肌细胞,采用全细胞膜片钳技术,研究不同浓度山莨菪碱对兔正常心室肌细胞跨膜钠离子通道电流（INa）、L-钙通道电流（ICa-L）及瞬间外向钾通道电流（Ito）的影响。结果山莨菪碱浓度为10nmol／L时,对INa无明显影响（P〉0．05）。山莨菪碱浓度为100nmol／L时,INa电流密度减少到（-33．25&#177;4．46）pA／pF,1000nmol／L时INa电流密度减少到（-29．32&#177;3．55）pA／pF,抑制率分别为22．3％和31．5％,与基础值比较,差异均有统计学意义（P〈0．01）。山莨菪碱浓度为10nmol／L时,对ICa-L无明显影响（P〉0．05）。山莨菪碱浓度为100nmol／L时,ICa-L．电流密度减少到（-2．15&#177;1．02）pA／pF,1000nmol／L时ICa-L电流密度减少到（-1．82&#177;0．86）pA／pF,抑制率分别为31．3％和41．8％,与基础值比较,差异均有统计学意义（P〈0．01）。山莨菪碱浓度为10nmol／L时,Ito电流密度由（17．41&#177;3．13）pA／pF减少到（16．13&#177;2．93）pA／pF,100nmol／L时减少到（15．11&#177;2．88）pA／pF,1000nmol／L时减少到（14．96&#177;2．82）pA／pF,抑制率分别为7．3％、13．2％和14．1％,均无统计学差异（P〉0．05）。结论山莨若碱对离体正常单个心室肌细胞INa和ICa-L具有剂量依赖性抑制作用。     

超声心动图评价血管紧张素受体拮抗剂对高血压病患者 左室舒张功能的影响

目的应用多普勒超声心动图评价血管紧张素II1型受体(AT1)拮抗剂对高血压病患者左室舒张功能的影响.方法分别于AT1拮抗剂(Losartan)服用前和服用12周后,对26例轻至中度原发性高血压病患者进行超声心动图检查.在心尖左心长轴切面上,用多普勒超声心动图测量二尖瓣口舒张早期峰值速度E、舒张晚期峰值速度A和E峰减速时间,并计算E/A比值;在左室长轴切面上,在二维超声心动图引导下用M型超声心动图测量舒张末期左室内径、室间隔和左室后壁厚度,并计算左室重量.结果服用Losartan 12周后,E/A比值(1.20±0.32)明显高于服用前(1.07±0.31,P＜0.0001),E峰减速时间从185±32 ms下降到164±29 ms(P=0.002);左室重量从220±53g减低为194±53g(P=0.003).结论AT1拮抗剂losartan具有改善高血压病患者左室舒张功能的作用.     

Evidence of Na Current Contribution to the Transient Outward Current in Cardiac Ventricular Myocytes

BACKGROUND: To study the transient outward current (I(to)) investigators often use sodium-free external solution to minimize the possible contamination of I(to) by sodium current. Removal of extracellular sodium creates reversal of sodium gradient and thus possibly contributing to I(to) mainly at positive potentials. METHODS AND RESULTS: To address this issue, whole-cell I(to) was recorded in sodium-free choline chloride and cobalt solutions, from rat ventricular myocytes known to exhibit a prominent I(to). Depolarizing pulse to 40 mV from -100 mV holding potential every 10 seconds elicited a fast activating and time-dependent inactivating components. The activation of I(to) was fast and complete within 10 ms at 40 mV, and the decay was rapid over the first 100 ms of the pulse and slower thereafter. External superfusion of the cell with 50 μM tetrodotoxin reversibly reduced I(to) amplitude by 25% from 1.47 +/- 0.2 to 1.1 +/- 0.3 nA (P <.04, n = 9). When sea anemone toxin (ATXII), known to selectively enhanced I(Na) by causing a delay in the inactivation gate, is applied to the cell, I(to) amplitude increased in a time- and dose-dependent manner (EC(50) =.86.4 nM). ATXII (100 nM) dramatically increased I(to) amplitude at all voltages between -20 and 60 mV (from 1.51 +/- 0.4 to 3.35 +/- 0.8 nA at 40 mV, P <.003, n = 12). Superfusion of cells with 5 mM 4-AP resulted in 82% reduction in I(to) amplitude at 40 mV (from 1.95 +/- 0.5 to 0.37 +/- 0.2 nA, P <.02, n = 8). Addition of ATXII to 4-AP containing solution increased peak I(to) by 965% (from 0.37+/-0.2 to 3.95 +/- 0.9, n = 8, P <.0003). However, in 11 other cells, addition of tetrodotoxin (50 μM) to the ATXII-containing solution blocked ATXII-induced outward current (from 3.51 +/- 0.64 nA to 1.60 +/- 0.17 nA, P <.05). The conductance (G(Ito)) was calculated by dividing peak I(to) by (Vm-E(K)), with an E(K) of -75 mV. G(Ito) was increased at all voltages (greater than -40 mV). Normalized G(Ito) was fitted by Boltzmann equation and ATXII did not significantly modify V(0.5) and k (from -20.5 +/- 3.9 to -17.0 +/- 3.5 mV for V(0.5), and 12.2 +/- 2.6 to 13.4 +/- 2.1 mV for k, n = 4). Also, atropine (1 μM) did not have any significant effect on I(to) (from 1.92 +/- 0.15 nA to 1.85 +/- 0.25 nA, n = 5). CONCLUSIONS: The results showed that, in sodium-free external solution I(to) is tetrodotoxin but not atropine sensitive. ATXII-induced I(to) increase is 4-aminopyridine insensitive but tetrodotoxin sensitive. These data suggest that outward Na current due to reversal of Na gradient in the absence of external Na contributes to I(to). Caution must be taken when studying kinetics and pharmacology of I(to) in external sodium-free solutions.